The treatment of tumor can be approached by several modes of therapy, including surgery, radiation, chemotherapy, or any combination of any of these treatments. Among them, chemotherapy is indispensable for inoperable or metastatic forms of cancer. Considering the diversity of tumors in terms of cell type, morphology, growth rate, and other cellular characteristics, the U.S. National Cancer Institute (NCI) has developed a xe2x80x9cdisease-orientedxe2x80x9d approach to anti-tumor activity screening. Boyd, M. R. (1989) In Principle of Practice of Oncology Devita, J. T., Hellman, S., and Rosenberg, S. A. (Eds.) Vol. 3, PPO Update, No. 10. This in vitro screening system is based on human tumor cell line panels consisting of approximately 60 cell lines of major human tumors (e.g., leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, or breast cancer), and serves as a tool for identifying compounds that possess anti-tumor activities.
Of particular interest are the anti-tumor compounds that function via one or more of the following four mechanisms: (1) inhibiting G2/M progression of the cell cycle, which might eventually induce the apoptosis in tumor cells (Yeung et al. (1999) Biochem. Biophys. Res. Com. 263: 398-404); (2) disturbing tubulin assembly/dissembly, which may inhibit the cell mitosis and induce the cell apoptosis (Panda et al. (1997) Proc. Natl. Acad. Sci. USA 94:10560-10564); (3) inhibiting endothelial cell proliferation and angiogenesis effect (Witte et al. (1998) Cancer Metastasis Rev. 17: 155-161; Prewett et al. (1999) Cancer Res. 59:5209-5218); or (4) regulating Ras protein-dependent signal transduction pathway (Hernandez-Alcoceba et al. (2000) Cell Mol. Life Sci. 57: 65-76; Buolamwini (1999) Cur. Opin. Che. Biol. 3: 500-509).
This invention is based in part on the discovery that piperazinedione compounds have anti-tumor activities, identified by NCI screening system, and function via one or more of the above-mentioned four mechanisms.
An aspect of the present invention relates to piperazinedione compounds of formula: 
Each of  and  independently, is a single bond or a double bond; A is H or CH(RaRb) when  is a single bond, or C(RaRb) when  is a double bond. Z is R3Oxe2x80x94(Ar)xe2x80x94B, in which B is CH(Rc) when  is a single bond, or C(Rc) when  is a double bond; Ar is heteroaryl; R3 is H, alkyl, aryl, heteroaryl, C(O)Rd, C(O)ORd, C(O)NRdRe, or SO2Rd; and both B and R3O can be substituted at any suitable position on Ar. Each of R1 and R2, independently, is H, C(O)Rd, C(O)ORd, C(O)NRdRe, or SO2Rd; and each of Ra, Rb, Rc, Rd, and Re, independently, is H, alkyl, aryl, heteroaryl, cyclyl, or heterocyclyl. Optionally, Ra and Rb taken together are cyclyl or heterocyclyl; and, also optionally, R1 and Ra or R1 and Rb taken together are cyclyl or heterocyclyl.
Referring to the above formula, a subset of the piperazinedione compounds of this invention is featured by that both  and  are double bonds. In these compounds, Ar is pyridyl linked to B at position 2, Rc is H, R3O is arylalkoxy linked to position 5 of pyridyl, both R1 and R2 are H, one of Ra and Rb is aryl or heteroaryl, and the other of Ra and Rb is H. Another subset of the piperazinedione compounds of this invention is featured by that both  and  are single bonds. In these compounds, Ar is pyridyl linked to B at position 2, Rc is H, R3O is arylalkoxy linked to position 5 of pyridyl, both R1 and R2 are H, one of Ra and Rb is H, aryl, or heteroaryl, and the other of Ra and Rb is H.
Alkyl, aryl, heteroaryl, cyclyl, and heterocyclyl mentioned herein include both substituted and unsubstituted moieties. The term xe2x80x9csubstitutedxe2x80x9d refers to one or more substituents (which may be the same or different), each replacing a hydrogen atom. Examples of substituents include, but are not limited to, halogen, hydroxyl, amino, alkylamino, arylamino, dialkylamino, diarylamino, cyano, nitro, mercapto, carbonyl, carbamido, carbamyl, carboxyl, thioureido, thiocyanato, sulfoamido, C1xcx9cC6 alkyl, C1xcx9cC6 alkenyl, C1xcx9cC6 alkoxy, aryl, heteroaryl, cyclyl, heterocyclyl, wherein alkyl, alkenyl, alkoxy, aryl, heteroaryl cyclyl, and heterocyclyl are optionally substituted with C1xcx9cC6 alkyl, aryl, heteroaryl, halogen, hydroxyl, amino, mercapto, cyano, or nitro. The term xe2x80x9carylxe2x80x9d refers to a hydrocarbon ring system having at least one aromatic ring. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and pyrenyl. The term xe2x80x9cheteroarylxe2x80x9d refers to a hydrocarbon ring system having at least one aromatic ring which contains at least one heteroatom such as O, N, or S. Examples of heteroaryl moieties include, but are not limited to, furyl, fluorenyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridinyl, pyrimidinyl, quinazolinyl, and indolyl.
Another aspect of the present invention relates to a pharmaceutical composition that contains a pharmaceutically acceptable carrier and an effective amount of at least one of the piperazinedione compounds described above.
A further aspect of this invention relates to a method for treating tumor (e.g., leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, or breast cancer). The method includes administering to a subject in need thereof an effective amount of the piperazinedione compound having the formula: 
Each of  and , independently, is a single bond or a double bond; A is H or CH(RaRb) when  is a single bond, or C(RaRb) when  is a double bond, Z is CH(RcRd) when  is a single bond, or C(RcRd) when  is a double bond; each of R1 and R2, independently, is H, C(O)Re, C(O)ORe, C(O)NReRf, or SO2Re; and each of Ra, Rb, Rc, Rd, Re, and Rf, independently, is H, alkyl, aryl, heteroaryl, cyclyl, or heterocyclyl, provided that one of Rc and Rd is aryl or heteroaryl. If  is a double bond,  is a single bond, and one of Rc and Rd is H, then the other of Rc and Rd is heteroaryl. Optionaly, Ra and Rb taken together are cyclyl or heterocyclyl; and, also optionally, R1 and Ra or R1 and Rb taken together are cyclyl or heterocyclyl.
Referring to the above formula, a subset of the just-described piperazinedione compounds is featured by that both  and  are double bonds. In these compounds, one of Rc and Rd is 2-pyridyl, the other of Rc and Rd is H, both R1 and R2 are H, one of Ra and Rb is aryl or heteroaryl, and the other of Ra and Rb is H. The 2-pyridyl can be further substituted with 5-arylalkoxy. Another subset of the piperazinedione compounds is featured by that both  and  are single bonds. In these compounds, one of Rc and Rd is 2-pyridyl, the other of Rc and Rd is H, both R1 and R2 are H, one of Ra and Rb is H, aryl, or heteroaryl, and the other of Ra and Rb is H.
Seven exemplary piperazinedione compounds are 3-[(5-benzyloxypyridin-2-yl)methylidene]-6-phenylmethylidene piperazine-2,5-dione, 3-[(5-benzyloxypyridin-2-yl)methylidene]-6-p-hydroxyphenylmethylidenepiperazine-2,5-dione, 3-[(5-benzyloxypyridin-2-yl)methylidene]-6-p-fluorophenylmethylidenepiperazine-2,5-dione, 3-[(5-benzyloxypyridin-2-yl)methylidene]-6-p-chlorophenylmethylidenepiperazine-2,5-dione, 3-[(5-benzyloxypyridin-2-yl)methylidene]-6-p-phenylmethoxy phenylmethylidenepiperazine-2,5-dione, 3-[(5-benzyloxypyridin-2-yl)methylidene]-6-[(thien-2-yl)methylidene]piperazine-2,5-dione, and 3,6-di [(5-benzyloxypyridin-2-yl)methyl]piperazine-2,5-dione. Their structures are shown below: 
The piperazinedione compounds described above include the compounds themselves, as well as their salts and their prodrugs, if applicable. Such salts, for example, can be formed between a positively charged substituent (e.g., amino) on a piperazinedione compound and an anion. Suitable anions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a negatively charged substituent (e.g., carboxylate) on a piperazinedione compound can form a salt with a cation. Suitable cations include, but are not limited to, sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as teteramethylammonium ion. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing piperazinedione compounds described above.
Also within the scope of this invention are a composition containing one or more of the piperazinedione compounds described above for use in treating tumor, and the use of such a composition for the manufacture of a medicament for the just-described use.
Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.
The piperazinedione compounds described above can be prepared by methods well known in the art, as well as by the synthetic routes disclosed herein. For example, one can react a piperazine-2,5-dione compound with a heteroaryl formaldehyde to produce an intermediate heteroaryl-methylidene-piperazine-2,5-dione. The intermediate can then be reduced to heteroaryl-methyl-piperazine-2,5-dione (a compound of this invention), or reacted with a ketone or another formaldehyde, followed by a base treatment, to produce a mixture of piperazinedione isomers, which are cis- or trans- or E- or Z-double bond isomeric forms. The desired isomeric product can be separated from others by high pressure liquid chromatography (HPLC). If preferred, proper functional groups can be introduced into the heteroaryl ring by further modifications. Alternatively, a desired reduced product can be obtained by reacting the product with a reducing agent.
Shown below is a scheme that depicts the synthesis of seventeen piperazinedione compounds. 
Details of synthesis of Compounds 1-17 are described in Examples 1-17, respectively. To prepare other piperazinedione compounds, the pyridinyl (shown in the above scheme) can be replaced by an aryl or another heteroaryl (e.g., furyl, pyrrolyl, imidazolyl, pyrimidinyl, or indolyl), and one of the two acetyl groups (Ac) on the piperazinedione ring (also shown in the above scheme) can be replaced by another substituent (e.g., carbonyl, carbamido, carbamyl, or carboxyl).
Note that the piperazinedione compounds contain at least two double bonds, and may further contain one or more asymmetric centers. Thus, they can occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z-double bond isomeric forms. All such isomeric forms are contemplated.
Also within the scope of this invention is a pharmaceutical composition that contains an effective amount of at least one piperazinedione compound of the present invention and a pharmaceutically acceptable carrier. Further, this invention covers a method of administering an effective amount of one or more of the piperazinedione compounds described in the xe2x80x9cSummaryxe2x80x9d section above to a subject in need of tumor treatment. The piperazinedione compounds can function via one or more of the above described action mechanisms, or via any other mechanism. xe2x80x9cAn effective amountxe2x80x9d refers to the amount of the compound which is required to confer a therapeutic effect on the treated subject. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described in Freireich et al., (1966) Cancer Chemother Rep 50: 219. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. An effective amount of the piperazinedione compounds can range from about 0.1 mg/Kg to about 50 mg/Kg. Effective doses will also vary, as recognized by those skilled in the art, depending on the types of tumors treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as use of other anti-tumor agents or radiation therapy.
To practice the method of the present invention, a piperazinedione compound-containing composition can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term xe2x80x9cparenteralxe2x80x9d as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
A sterile injectable composition, for example, a sterile injectable aqueous or oleaginous suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.
A composition for oral administration can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added. A nasal aerosol or inhalation composition can be prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. A piperazinedione compound-containing composition can also be administered in the form of suppositories for rectal administration.
The carrier in the pharmaceutical composition must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the active ingredient of the formulation (and preferably, capable of stabilizing it) and not deleterious to the subject to be treated. For example, solubilizing agents such as cyclodextrins, which form specific, more soluble complexes with the piperazinedione compounds, or one or more solubilizing agents, can be utilized as pharmaceutical excipients for delivery of the piperazinedione compounds. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and DandC Yellow #10.
The piperazinedione compounds can be preliminarily screened for their efficacy in treating cancer by one or more of the following in vitro assays.
One assay is based on the NCI screening system, which consists of approximately 60 cell lines of major human tumors. See Monks, et al. (1991) JNCI, J Natl. Cancer Inst. 83: 757-766; Alley, et al. (1988) Cancer Res. 48: 589-601; Shoemaker, et al. (1988) Proc. Clin. Biol. Res. 276: 265-286; and Stinson, et al. (1989) Proc. Am. Assoc. Cancer Res. 30: 613. Briefly, a cell suspension that is diluted according to the particular cell type and the expected target cell density (5,000-40,000 cells per well based on cell growth characteristics) is added (100 xcexcL) into a 96-well microtiter plate. A pre-incubation is preformed at 37xc2x0 C. for 24 hr. Dilutions at twice of an intended test concentration are added at time zero in 100 xcexcL aliquots to each well of the microtiter plate. Usually, a test compound is evaluated at five 10-fold dilutions. In a routine testing, the highest concentration of the test compound is 10xe2x88x924 M. Incubations are performed for 48 hr in 5% CO2 atmosphere and 100% humidity. The cells are assayed by using the sulforhodamine B assay described by Rubinstein, et al. (1990, JNCI, J Natl. Cancer Inst. 82: 1113-1118) and Skehan, et al. (1990, JNCI, J. Natl. Cancer Inst. 82: 1107-1112). A plate reader is used to read the optical densities and a microcomputer processes the optical densities into the special concentration parameters. The NCI has renamed an IC50 value, the concentration that causes 50% growth inhibition, a GI50 value to emphasize the correction for the cell counted at time zero; thus, the GI50 measures the growth inhibitory power of the test compound. See Boyd, et al. (1992) In Cytotoxic Anticancer Drugs: Models and Concepts for Drug Discovery and Development; Vleriote, F. A.; Corbett, T. H.; Baker, L. H. (Eds.); Kluwer Academic: Hingham, Mass., pp 11-34.
In another assay, a piperazinedione compound is tested for its cytotoxicity on PC-3 cells (a prostate cancer cell line). More specifically, cells are incubated with a test compound in a serum-free medium for 24 hr. The cytotoxic effect can be determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method described in Boyd (In Principle of Practice of Oncology Devita, J. T., Hellman, S., and Rosenberg, S. A. (Eds.) Vol. 3, PPO Update, No. 10, 1989).
Another in vitro assay can be used to evaluate the efficiency of a piperazinedione compound in arresting the cell cycle progression. More specifically, a test piperazinedione compound is added to PC-3 cells in a concentration-dependent manner using propidium iodide-stained flow cytometric assessment. The cell population of sub-G0/G1, G0/G1, S, and G2/M phase is then determined. In addition, the effect of a piperazinedione compound on the Ras activity can be examined to determine its regulation of Ras protein-dependent signal transduction pathway.
The anti-tumor activity of a piperazinedione compound can be further assessed by an in vivo animal model. Using SCID mice as the model, PC-3 cells are subcutaneously injected into the mice to develop a prostate tumor. The anti-tumor activity of a piperazinedione compound is determined after treatment. Additionally, the anti-tumor activity of a piperazinedione compound can also be evaluated using in vivo anti-angiogenesis testing. For example, nude mice can be used to test the effect of a piperazinedione compound on bFGF-induced angiogenesis. A matrigel with bFGF or vscular endothelial growth factor (VEGF) is subcutaneously injected into a mouse with concurrent intraperitoneal administration of a piperazinedione compound. After several days of incubation, the matrigel is cut down for examination of angiogenesis.
Without further elaboration, it is believed that the above description has adequately enabled the present invention. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All of the publications cited herein are hereby incorporated by reference in their entirety.